A fundamental area of inquiry in biology is the analysis of interactions between proteins. Proteins are complex macromolecules made up of covalently linked chains of amino acids. Each protein assumes a unique three dimensional shape determined principally by its sequence of amino acids. Many proteins consist of smaller units termed domains, which are continuous stretches of amino acids able to fold independently from the rest of the protein. Some of the important forms of proteins are enzymes, polypeptide hormones, receptors, nutrient transporters, structural components of the cell, hemoglobins, antibodies, nucleoproteins, and components of viruses.
Protein-protein interactions enable two or more proteins to associate. A large number of non-covalent bonds form between the proteins when two protein surfaces are precisely matched. These bonds account for the specificity of recognition. Protein-protein interactions are involved, for example, in the assembly of enzyme subunits; in antigen-antibody reactions; in forming the supramolecular structures of ribosomes, filaments, and viruses; in transport; and in the interaction of receptors on a cell with growth factors and hormones. Products of oncogenes can give rise to neoplastic transformation through protein-protein interactions. For example, some oncogenes encode protein kinases whose enzymatic activity on cellular target proteins leads to the cancerous state. Other examples of protein-protein interaction include when a virus infects a cell by recognizing a polypeptide receptor on the surface and when platelets aggregate during thrombosis.
Protein-protein interactions have been generally studied in the past using biochemical techniques such as cross-linking, co-immunoprecipitation and co-fractionation by chromatography. A disadvantage of these techniques is that interacting proteins often exist in very low abundance and are, therefore, difficult to detect. Another major disadvantage is that these biochemical techniques involve only the proteins, not the genes encoding them. When an interaction is detected using biochemical methods, the newly identified protein often must be painstakingly isolated and then sequenced to enable the gene encoding it to be obtained. Another disadvantage is that these methods do not immediately provide information about which domains of the interacting proteins are involved in the interaction.
To alleviate the problems associated with the biochemical characterization of protein-protein interactions, genetic systems have been invented that are capable of rapidly detecting which proteins interact with a known protein, determining which domains of the proteins interact, and providing the genes for the newly identified interacting proteins. One such system is the yeast two-hybrid system wherein two proteins are expressed in yeast: one protein of interest fused to a DNA-binding domain and the other protein of interest fused to a transcriptional activation domain (Fields et al. (1989) Nature 340:245; Gyuris et al. (1993) Cell 75:791; Harper et al. (1993) Cell 75:805; Serrano et al. (1993) Nature 366:704; and Hannon et al. (1993) Genes & Dev. 7:2378). If the proteins interact, they activate transcription of a reporter gene that contains a binding site for the DNA-binding protein.
Although the development of genetic systems that utilize direct activation of a reporter gene, such as the yeast two-hybrid systems, has greatly facilitated the study of protein-protein interactions, many problems remain to be solved. For instance, the yeast two-hybrid systems rely on interactions between the two proteins in the nucleus of the cell. Accordingly, yeast two-hybrid systems are not useful for the study of integral membrane protein interactions and cannot be used to test cell membrane impermeate drugs. Furthermore, the study of protein-protein interactions wherein one of the proteins is itself a transcriptional activator often results in the transcription of the reporter gene without interaction between the two proteins under study. Lastly, the yeast two-hybrid systems require that both proteins under study be expressed as fusion proteins resulting in the possible loss of function.